Alpha-1-antitrypsin (AAT) deficiency is the second most common monogenic lung disease in man, accounting for approximately 3% of all early deaths due to obstructive pulmonary disease. AAT protein is normally produced in the liver, secreted into the serum and circulated to the lung where it protects the fine supporting network of elastin fibers from degradation by neutrophil elastase. Current therapy for AAT deficiency includes avoidance of cigarette smoke exposure and weekly intravenous infusions of recombinant human AAT (hAAT) protein. Attempts to devise gene therapy strategies to replace AAT either in the lung itself or within any of a number of other tissues which are capable of AAT secretion have been limited by the short duration of expression from some vectors and by the relatively high circulating levels of AAT which is required for therapeutic effect. Methods of gene therapy have been described in U.S. Pat. No. 5,399,346.
It has recently been demonstrated that adeno-associated virus (AAV) vectors are capable of stable in vivo expression and may be less immunogenic than other viral vectors (Flotte et al., 1996; Xiao et al., 1996; Kessler et al., 1996; Jooss et al., 1998). AAV is a non-pathogenic human parvovirus whose life cycle naturally includes a mechanism for long-term latency. In the case of wild-type AAV (wtAAV), this persistence is due to site-specific integration into a site on human chromosome 19 (the AAVSI site) in the majority of cells (Kotin et al., 1990), whereas with recombinant AAV (rAAV) vectors, persistence appears to be due to a combination of episomal persistence and integration into non-chromosome 19 locations (Afione et al., 1996; Kearns et al., 1996). Recombinant AAV latency also differs from that of wtAAV in that wtAAV is rapidly converted to double-stranded DNA in the absence of helper virus (e.g., adenovirus) infection, while with rAAV leading strand synthesis is delayed in the absence of helper virus (Fisher et al., 1996; Ferrari et al., 1996). U.S. Pat. No. 5,658,785 describes adeno-associated virus vectors and methods for gene transfer to cells.
Kessler et al. (1996) demonstrated that murine skeletal myofibers transduced by an rAAV vector were capable of sustained secretion of biologically active human erythropoietin (hEpo), apparently without eliciting a significant immune response against the secreted hEpo. See also U.S. Pat. No. 5,858,351 issued to Podsakoff et al. Likewise, Murphy et al. (1997) have observed the expression and secretion of sustained levels of leptin in ob/ob mice after AAV muscle transduction. Brantly et al. (U.S. Pat. No. 5,439,824) disclose methods for increasing expression of AAT using vectors comprising intron II of the human AAT gene. However, the level of leptin expression observed was only in the range of 2 to 5 ng/ml. Therapy for AAT deficiency requires serum levels of at least about 800 xcexcg/ml. Thus, there remains a need in the art for a means of providing therapeutically beneficial levels of a protein to a person in need of such treatment.
The subject invention concerns materials and methods for gene therapy. One aspect of the invention pertains to vectors which can be used to provide genetic therapy in animals or humans having a genetic disorder where relatively high levels of expression of a protein is required to treat the disorder. The vectors of the invention are based on adeno-associated virus (AAV). The vectors are designed to provide high levels of expression of heterologous DNA contained in the vector. In one embodiment, the vectors comprise AAV inverted terminal repeat sequences and constitutive or regulatable promoters for driving high levels of gene expression. The subject invention also pertains to methods for treating animals or humans in need of gene therapy, e.g., to correct a genetic deficiency disorder.